Hello fellow colleagues and scientists,
we want to determine the purity of our Protein A purified human antibodies via SDS-PAGE. We observe only one band at 150kDa in the non-reduced SDS-PAGE as we would expect it.
In the reduced SDS-PAGE on the other hand we see bands for all the different antibody fragments, which hints to an incomplete reduction of the disulfide bridges between the fragments. The optimal PAGE would only show the 50kDa and 25kDa bands for the heavy and light chains and nothing else.
As 4x sample buffer we use:
glycerol (30%), tris-base (1M, pH = 6,8 with HCl) (25%), SDS (5%), 2-mercaptoethanol (20%), DTT (400mM), bromophenol blue (0,02%).
Which gives a final reducing agent concentration of 5% 2-mercaptoethanol and 100mM DTT.
We heat the samples for 10 Minutes at 90°C before loading them to the gel. The gels were silver-stained. See them attached.
We also tried alkylation with iodoaceteamide and NEM after and during the heating step, which didn't make a difference at all.
We would be thankful for any hints or suggestions to obtain only the two fully reduced bands of the heavy and light chains from the antibody.
Thanks a lot!
I would suggest using TCEP instead of DTT or 2-BME, since TCEP reacts irreversibly unlike DTT and 2-BME
I forgot to mention that we already tried TCEP with no different result. Sorry.
Hi Marco,
In your gel for the (-) reduced you see two bands above you antibody band. Maybe this could be some of the bands you see in the + reduced (they are absent here). One "problem" with the silver stain is that it is so sensitive that you see very small quantities. You could try and identify the faint bands in the (+) reduced with mass spectrometry.
Another thing you could try (I would recommend) is to ammonium precipitate a small portion of your already purified protein A antibody. This should remove the bands above your antibody band. Or maybe even easier would be to polish your antibody on a gel filtration column (superdex 200 or superose 6) and then run a silver stain gel.
Please share the results with us when you have some.
Kind regards,
Simon
I would add a chaotrope (urea is compatible with SDS-PAGE) to help denature the protein to allow more efficient reduction of the disulfide bond.
An Update:
We tried to use Urea as an additive as suggested by Vince Murphy
Unfortunately the results were the same.
As a next step we try to use way less reducing agents.
First, Ab are evolved to perform in extreme environments, and so they are often especially difficult to reduce for PAGE. If you extensively reduce and alkylate in the presence of chaotrope, before you run out the sample on a reducing gel, then that's simply the best result you can get for that particular Ab.
Second, while PAGE is good for detection of impurities, it's not a quantitative comparison, unless you run standard curves on the Ab and the impurity. Most people prefer LC for this task.
We couldn't see a big difference while using less reduction agent, besides a bit smoother bands.
@John Carter That's a very good explanation and I haven't thought of that yet. It makes even more sense, if we consider that artificial antibodies like Herceptin or Avastin are more fragile and therefore produce two clear bands without wide-spread patterns.
Thanks for the advice.
So to conclude:
Natural occurring antibodies from whole serum or plasma are compared to artificial antibodies more resistant to heat, chemical stress or modification. That's why you either can't reduce them completely or on the other hand create unspecific fragments while using too much reducing agents.
Correct me if I'm wrong.
Maybe sometimes, at least. In my experience, engineered antibodies are more fragile than others. I think you're saying that you're isolating Ab from a polyclonal source. Polyclonal antibodies include various populations of tough and fragile species.
Dear John,
Are there scoentific proof for your statement: “Polyclonal antibodies include various populations of tough and fragile species.“
Kind regards,
Simon
The word "polyclonal" defines pAb as heterogenous, which is what I mean when I write that they "include various populations". For example, the functionality of pAb typically changes over time, so that a serum sample taken one week exhibits a significantly different titer from a sample taken in the next week. Immunological paradigm suggests that at least some of this change is due to affinity maturation, meaning in vivo selection of different molecular species according to functionality.
Thus we might think of pAb as mixtures of many mAb, and some mAb are hardier than others. For example, many mAb survive acid elution during affinity purification, while others substantially lose binding activity. Some of these perform OK after chaotrope elution, while others flunk out of screening because of poor or irreproducible yield in purification, no matter what you try.
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